Parasitic nematode (PN) infections remain a major threat to human health worldwide, with more than 1 billion people infected. Children, pregnant women, and the elderly are particularly susceptible to morbidity from nematode infection. Control strategies are restricted to periodic de-worming of infected individuals, which is limited by rapid re-infection rates and the development of drug resistant worm populations. There are no vaccines available for PN infections in humans. Development of new drugs and vaccines will require a better understanding of PN biology, particularly the infective process and the host's immune response to infection. The requirement of an obligate host and the lack of good animal models have limited investigations into mechanisms by which PNs infect and usurp the host immune response. While the free living nematode Caenorhabditis elegans is an excellent model for nematode development, it is not a parasite. The insect parasitic nematode Heterorhabditis bacteriophora (Hb) offers potential as a tractable model of PN infection that has many of the advantages of C. elegans, including easy culture and manipulation of all life history stages. However, many of the powerful genetic tools, like transgenesis and RNA interference, have not been fully developed in Hb, restricting its use as a widespread model. We propose to develop these genetic tools in Hb with a focus on targeting the infective juvenile and early parasitic stages in order to investigate infection mechanisms. We will take two parallel but independent approaches to develop these tools that will provide a foundation for future investigation into the mechanism of PN infection. In Aim 1, we propose to develop a transfection protocol for Hb via germ-line microinjection of a piggybac retrotransposon-based integrating vector to introduce the reporter transgenes into Hb chromosomes. In Aim 2, we will develop a robust RNAi protocol for 2nd generation Hb hermaphrodites. By focusing on 2nd generation hermaphrodites we are ideally positioned to probe the biology of PN infection, as the progeny of this stage are the infective stage. Development of methods for gene knock down (reverse genetics) and transfection in this novel model nematode would represent a significant advance for PN research, and will allow investigation of PN gene function during infection for the first time.